The present invention relates to the field of hematology, and in particular, to assays for determining the number or presence of the formed elements of blood such as red blood cells, white blood cells, platelets and reticulocytes. More specifically, the invention relates to control compositions used in performing such assays, and to processes for preparing, and methods of using such control compositions.
The field of hematology involves the study of blood, including the discrete cell populations that make up the blood and blood forming organs. The ability to reliably and accurately distinguish and count cells is one important tool in this field. Clinical significance can be attributed to abnormal levels, in both relative and absolute terms, of most cell populations.
The study of the dynamics of blood cell production and destruction depends upon the enumeration of new cells being delivered to the circulation per unit time. New immature red blood cells (known as reticulocytes) are easily identified and can be quantified as a percentage of total red blood cells (xe2x80x9cRBCsxe2x80x9d).
A reticulocyte is created during the final stages of erythroid development, resulting from the enucleation of RNA-rich, orthochromatic normoblasts. The feature of reticulocytes most frequently used to distinguish them from mature RBCs is their stainable RNA. Reticulocytes most recently released into blood from the bone marrow compartment contain the highest levels of cellular RNA. As reticulocytes mature, the cells gradually lose RNA, and this biochemical change can be used to determine not only the presence of reticulocytes, but also can serve as an indicator of reticulocyte maturational stages. Within 1 to 2 days from being released from the bone marrow compartment, the reticulocytes lose RNA to a point where the cells become indistinguishable from mature RBCs.
As a result of the foregoing, reticulocytes can be distinguished from mature red blood cells by the presence in reticulocytes of cellular RNA. Other less prevalent methods of identifying reticulocytes involve staining of mitochondria, ribosomes, and other cytoplasmic organelles (with so-called supravital dyes, such as new methylene blue, brilliant cresyl blue, and acridine orange), as well as methods involving recognition of cell surface markers unique to reticulocytes. The reticulocyte percentage is then multiplied by the red cell count in order to provide the number of reticulocytes per microliter.
Reticulocytes require 3 to 4 days to mature into non-stainable red blood cells, with about 2 to 3 days of this period being spent in the bone marrow and about 1 to 2 days being spent in the peripheral blood. The reticulocyte count can be clinically significant, if accurately measured. Levels approaching 3% of the total number of circulating red cells may indicate increased marrow activity, e.g., when blood synthesis is stimulated with erythropoietin. In contrast, levels below about 0.5% can be an indication of bone marrow incompetence. Hence, a reticulocyte count is an effective measure of marrow erythroid output.
Many approaches have been described for determining absolute and/or relative reticulocyte levels. Although such approaches have traditionally been performed manually, automated procedures are being used with increasing frequency. An increasing number of manufacturers of automated cell counting instruments have added the ability to count reticulocytes to their systems. Additionally, some instruments provide the ability to measure the relative maturity of reticulocytes, which is inversely proportional to the amount of stainable matter (e.g., RNA) within the cell. This indicates the relative age of the reticulocyte leaving the bone marrow. Such values may be referred to as the immature reticulocyte fraction (xe2x80x9cIRFxe2x80x9d), the reticulocyte maturation index (xe2x80x9cRMIxe2x80x9d), and other such terms commonly used to describe this measurement.
The newest of the cell counting instruments include the reticulocyte count, complete blood count (xe2x80x9cCBCxe2x80x9d), and the white blood cell (xe2x80x9cWBCxe2x80x9d) differential count. However, current controls for such instruments are separate products (i.e., separate CBC/WBC differential control and separate reticulocyte control) that must be run on these instruments in two distinct steps. In general, a CBC plus differential control critiques the ability of a diagnostic instrument to correctly determine the total white cell count and the percentage of the five major classes of white blood cells (leukocytes) commonly found in circulation which comprise this total, namely, lymphocytes, neutrophils, monocytes, eosinophils, and basophils, as well as total RBCs and platelets in the sample. When using these latest counting instruments, the CBC/WBC differential control is run and recovered values for this control are compared with expected values. Thereafter, the reticulocyte only control is run and recoveries are compared with the expected values.
Thus, the present inventors believe it would be advantageous to provide a single hematology control that allows measurement of the formed elements of blood, including reticulocytes, red blood cells, white blood cells, (and subpopulations thereof) and platelets. Such a unitary control would avoid the necessity of performing separate counts to obtain values for the components of whole blood.
Each reticulocyte counting method relies upon a standardized reticulocyte control to assess the accuracy and reliability of the results. Reticulocyte-only controls have been provided in a number of different forms. Human blood is generally considered a poor source of reticulocytes for controls, because it is typically too low in reticulocyte count to be useful for the preparation of a wide range of control levels.
A control composition known as Retic-C(trademark) is available in three control levels from Beckman/Coulter Corporation (Miami, Fla.). This product includes avian red blood cells as reticulocyte analogues. Avian RBCs are significantly larger than human reticulocytes and, in contrast to reticulocytes, also contain a nucleus. Because these cells are not true reticulocytes, they do not stain in the manner common to reticulocytes. As a result, the composition is limited to use on Coulter automated instruments such as the Coulter STKS(trademark).
Another reticulocyte-only control composition is a reticulocyte analogue product available as the Retic-CHEX(trademark) from Streck Laboratories (Omaha, Nebr.). This control contains a reticulocyte analogue that suffers from poor staining intensity and displays characteristics only somewhat like that of true reticulocytes. Yet other control compositions, both of which are manufactured by Streck Laboratories, include the Test Point(trademark) product (available in 2% and 5% levels for use with Bayer instruments), and a 2% and 5% level control available for instruments manufactured by Sysmex. Both compositions are limited in that they provide only two reticulocyte levels, with the upper level being significantly lower than desired.
The present inventors previously described a reticulocyte-only control composition comprising stabilized, maturation-arrested porcine reticulocytes in a red blood cell base, wherein the red blood cell base comprises mature erythrocytes. The present inventors have also described methods of preparing and methods of using such a reticulocyte control. See U.S. Pat. Nos. 5,858,789 and 5,736,402, and 5,945,340 (all of which are commonly owned by the assignee of the present application, and the disclosure of each is incorporated by reference). Unlike the controls described above, this reticulocyte control contains true reticulocytes, obtained from pigs. By avoiding the use of reticulocyte analogs as described above in connection with other commercially available products, this control provides a more accurate reticulocyte count. As described in the cited patents, mature erythrocytes for the control can be provided from porcine, human, or other mammalian sources.
The above reticulocyte controls provide stand-alone controls that measure the levels of only reticulocytes in a sample. These controls can be used in the reticulocyte step of the two-stage whole blood analysis described above. However, it would be particularly advantageous if a single control were capable of identifying and quantitating the various parameters of a whole blood sample, including total RBC, WBC, platelet and reticulocyte content. Such a whole blood control would more closely resemble a whole blood sample of a patient, which would in turn allow a more complete analysis of a sample.
Previously, reticulocytes had to be measured in a separate mode or by a completely separate analysis, as discussed above. This separate analysis was necessary because the diagnostic instruments were not capable of measuring the CBC/WBC differential as well as reticulocytes in the same sample run. Although such CBC/WBC differential controls typically contained a small amount of residual reticulocytes, such reticulocytes were simply present within the red blood cell source used to prepare the control, and were not intentionally added to the control in a significant amount. Such CBC/WBC differential controls generally contained, at most, 1% or less reticulocytes. In addition, the reticulocyte count and maturation level in such samples were not carefully controlled, making such samples inappropriate for use as reticulocyte controls.
Some currently available reticulocyte controls have significant practical limitations in wide-scale application as whole blood controls. If the control includes cells that simulate reticulocytes and contain a nucleus (e.g., avian RBCs), for example, these cells may adversely affect the total WBC count, and may possibly interfere with the WBC differential of the control. The presence of a nucleus, as well as the size of the reticulocyte component of the control, can interfere with the identification of other nucleated cells in the sample.
There do not appear to be any control compositions available today that provide an optimal combination of stability, true reticulocyte appearance and stain characteristics, and wide utility with most of the presently available assay techniques, that are further incorporated into a complete hematology control matrix which most resembles a patient sample. Hence, there is a need for a control composition providing an optimal combination of these properties, which can be used with semi-automated and automated procedures.
The present invention provides a control composition comprising a predetermined concentration of stabilized reticulocytes in a complete hematology control matrix. In one embodiment, the complete hematology control matrix comprises a complete blood count base that includes mature erythiocytes (red blood cells), stabilized white blood cells or white blood cell analogs, and stabilized platelets or platelet analogs.
In a preferred embodiment, the reticulocytes are maturation-arrested by treatment with a reticulocyte storage buffer which optimally includes an inhibitor of eucaryotic protein synthesis or metabolism. In this embodiment, the relative maturity of the reticulocytes can be controlled to provide a desired maturity profile within the control. Particularly preferred inhibitors are selected from cycloheximide and rifampicin.
An additional embodiment comprises a ready-to-use kit which includes a number of control compositions wherein one or more of the control compositions include reticulocytes, mature red blood cells, stabilized white blood cells (or analogs thereof), and stabilized platelets (or analogs thereof). In one preferred version of this embodiment, the reticulocytes are provided at concentrations from about 0.5% to about 25%. More preferably, reticulocytes are provided at concentrations from about 0.5% to about 15%. In an optimal embodiment, the reticulocytes are provided at concentrations from about 1% to about 12%.
The invention also encompasses a method of preparing a control composition that includes the steps of harvesting an enriched population of reticulocytes; treating the reticulocytes with a suitable medium to arrest further maturation; stabilizing the harvested, arrested reticulocytes; and preparing a control composition that includes a pre-determined concentration of these stabilized, maturation-arrested reticulocytes, RBCs, WBCs (or analogs thereof), and platelets (or analogs thereof). The process of harvesting reticulocytes can involve a differential sedimentation process described in more detail below. Optimally, the treatment of the reticulocytes with a suitable medium generates a maturity profile, so that the relative maturity of the reticulocytes to be used in the control can be controlled.
The resulting control composition provides an optimal combination of such properties as true reticulocyte appearance and stain characteristics; stability; uniformity; and wide utility as a control with most, if not all, presently available assay techniques. Additionally, the present invention provides a unitary control that more closely resembles a patient sample and allows measurement of reticulocyte levels and relative maturity of reticulocytes.
According to the present invention, the addition of the reticulocyte count to the CBC/WBC differential yields a more complete assessment of erythropoietic activity. A CBC/WBC differential plus reticulocyte control provides a hematology control that more closely simulates a human whole blood sample. Further, the analysis of the relative RNA content of circulating reticulocytes yields additional information regarding the rate of erythrocyte regeneration. The ability to reproducibly control the maturity of the reticulocyte component of the control also enables assessment of the analyzer""s ability to detect recently synthesized erythroid cells entering the circulation. The present invention can be used with a variety of analytical methods, for example, semi-automated and automated methods to analyze whole blood samples in a single step.